The present invention relates to novel monomeric fusion proteins derived from human GAG binding proteins such as chemokines with increased glycosaminoglycan (GAG) binding affinity and knocked-out or reduced G-protein coupled receptor (GPCR) activity compared to wild type GAG binding proteins fused to human serum albumin, which are highly selectively competitive and are of increased bioavailability, and to their use for prevention or treatment of pathological cell movement as in metastasis.
Chemokines are well-known key players in the immune system and in the process of angiogenesis and are also involved in pathological conditions like cancer. The interaction with cell-surface heparan sulfate proteoglycans is essential for their signaling via G-protein coupled receptors.
Chemokines stand for a large group of small cytokines. Their name is the result of their ability to induce chemotaxis or the directed movement of cells through a concentration gradient: chemotactic cytokines. The first chemokine to be characterized was Interleukin 8 in 1987. Nowadays there are about 50 known ligands, 18 standard receptors and 5 atypical receptors of the human chemokine family. In their monomeric form their molecular weight of the ligands ranges from 8-12 kDa, the receptors are about 40 kDa. It was found that chemokine genes tend to form specific clusters on certain chromosomal sites.
All chemokines, with the exception of lymphotactin and fraktaline/neurotactin which are members of the C and CX3C chemokine subfamily, respectively, have four cysteines in conserved positions and can be divided into the CXC or α-chemokine and the CC or β-chemokine subfamilies on the basis of the presence or absence, respectively, of an amino acid between the two cysteines within the N-terminus. Chemokines are small secreted proteins that function as intercellular messengers to orchestrate activation and migration of specific types of leukocytes from the lumen of blood vessels into tissues (Baggiolini M., J. Int. Med. 250, 91-104 (2001)). This event is mediated by the interaction of chemokines with seven transmembrane G-protein-coupled receptors (GPCRs) on the surface of target cells. Such interaction occurs in vivo under flow conditions. Therefore, the establishment of a local concentration gradient is required and ensured by the interaction of chemokines with cell surface glycosaminoglycans (GAGs). Chemokines have two major sites of interaction with their receptors, one in the N-terminal domain which functions as a triggering domain, and the other within the exposed loop after the second cysteine, which functions as a docking domain (Gupta S. K. et al., Proc. Natl. Acad. Sci., USA, 92, (17), 7799-7803 (1995)). The GAG binding sites of chemokines comprise clusters of basic amino acids spatially distinct (Ali S. et al., Biochem. J. 358, 737-745 (2001)). Some chemokines, such as RANTES, have the BBXB motif in the 40 s loop as major GAG binding site; IL-8 interacts with GAGs through the C-terminal α-helix and Lys 20 in the proximal N-loop. Other chemokines, such as MCP-1, show a significant overlap between the residues that comprise the receptor binding and the GAG binding site (Lau E. K. et al., J. Biol. Chem., 279 (21), 22294-22305 (2004)).
IL (interleukin)-8 (CXCL8, CXC chemokine ligand 8)) is an 8 kDa CXC-chemokine that attracts neutrophils to sites of inflammation when immobilized on endothelial GAG chains in the vasculature. The subsequent binding of the chemokine to the neutrophil GPCRs (G-protein coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, fully activates the already slowed down (selectin-mediated ‘rolling’) neutrophil and leads to firm adhesion and subsequent transmigration through the blood vessel endothelium into the tissue. The three-dimensional solution structure of CXCL8 shows a dimer with two symmetry-related, antiparallel α-helices, which lie on top of six-stranded antiparallel β-sheets derived from two three-stranded Greek keys, one from each monomer unit. Despite its small size, CXCL8 exhibits discrete but connected structural domains by which the chemokine interacts with its two biological receptors: with the traditional GPCRs CXCR1 and CXCR2 on the one hand and with GAG co-receptors on the other hand (Falsone A. et al., Biosci. Rep., 2013, 33(5), e00068).
In the context of the chemokine-β family of cytokines, monocyte chemoattractant protein-1 (MCP-1) is a monocyte and lymphocyte-specific chemoattractant and activator found in a variety of diseases that feature a monocyte-rich inflammatory component, such as atherosclerosis (Nelken N. A. et al., J. Clin. Invest. 88, 1121-1127 (1991); Yla-Herttuala, S., Proc. Natl. Acad. Sci USA 88, 5252-5256 (1991), rheumatoid arthritis (Koch A. E. et al., J. Clin. Invest. 90, 772-779 (1992); Hosaka S. et al., Clin. Exp. Immunol. 97(3), 451-457 (1994), Robinson E. et al., Clin. Exp. Immunol. 101(3), 398-407 (1995)), inflammatory bowel disease (MacDermott R. P. et al., J. Clin. Immunol. 19, 266-272 (1999)) and congestive heart failure (Aukrust P., et al., Circulation 97, 1136-1143 (1998), Hohensinner P. J. et al., FEBS Letters 580, 3532-3538 (2006)). Crucially, knockout mice that lack MCP-1 or its receptor CCR2, are unable to recruit monocytes and T-cells to inflammatory lesions (Grewal I. S. et al., J. Immunol. 159 (1), 401-408 (1997); Boring L. et al., J. Biol. Chem. 271 (13), 7551-7558 (1996); Kuziel W. A., et al., Proc. Natl. Acad. Sci. USA 94 (22), 12053-8 (1997); Lu B., et al., J. Exp. Med. 187 (4), 601-8 (1998)); furthermore, treatment with MCP-1 neutralizing antibodies or other biological antagonists can reduce inflammation in several animal models (Lukacs N. W. et al., J. Immunol., 158 (9), 4398-4404 (1997); Flory C. M. et al., 1. Lab. Invest. 69 (4), 396-404 (1993); Gong J. H., et al., J. Exp. Med. 186 (1), 131-7 (1997); Zisman D. A. et al., J. Clin. Invest. 99 (12), 2832-6 (1997)). Finally, LDL-receptor/MCP-1-deficient and apoB-transgenic/MCP-1-deficient mice show considerably less lipid deposition and macrophage accumulation throughout their aortas compared to the WT MCP-1 strains (Alcami A. et al., J. Immunol. 160 (2), 624-33 (1998); Gosling J. et al., J. Clin. Invest. 103 (6), 773-8 (1999)).
Piccinini et al. have shown the effect of a limited number of site-directed MCP-1 mutants on enhanced glycosaminoglycan binding (J Biol Chem. 2010 Jan. 22). Liehn et al. have shown that increasing the GAG binding affinity has a therapeutic effect in murine models of myocardiac infarction and restenosis (J. Am. Coll. Cardiol., 23:56(22):1847-57, 2010).
Proudfoot et al. (Proc. Natl. Acad. Sci., 100, 4, 2003, 1885-1890) investigated the effect of mutations in the GAG binding sites of chemokines, amongst others of MCP-1. The specific mutant (18AA19)-MCP-1 shows only residual affinity for heparin.
US2003/0162737 discloses an antagonistic MCP-1 mutein for the treatment of pulmonary hypertension. Said MCP-1 mutein comprises several deletions at the N-terminus of the protein, up to deletion of N-terminal amino acids 1-10 or 2-8. Further the mutein can comprise a modification at amino acid positions 22 or 24.
Steitz S. et al. (FEBS Letters, 430, 3, 1998, 158-164) investigated the role of N-terminal modifications on receptor binding. MCP-1 mutants comprising substitutions of amino acid positions 13 and 18 were disclosed. Y13A showed a dramatic loss in function to induce THP-1 chemotaxis.
Lubkowski J. et al. (Nature Structural Biology, 4, 1, 1997, 64.69) investigated the x-ray crystal structure of recombinant human MCP-1. The N-terminus of the protein was modified and its effect on activity was measured. It was shown that modification specifically at positions 10 and 13 lowered the activity of MCP-1 and had an effect on the dimer stabilization. An impaired chemotactic activity of the mutants suggested a functional significance for Tyr28, Arg 29, Arg30 and Asp68. It was noted that charged amino acids (Arg, Asp) destabilize an alternate dimer and that the introduction of uncharged residues can significantly increase stability.
WO2010086426A1 describes modified MCP-1 mutant proteins with increased GAG binding affinity.
US20110280873A1 reports the development of MCP-1 Ig fusion polypeptides for treating diseases.
US20070036750A1 also discloses MCP-1 fusion proteins linked to immunoglobulins and their use to treat medical disorders.
WO2008074047 describes chemokine proteins fused to a chaperone peptide, e.g. heat shock proteins.
WO2005054285A1 describes chemokine mutants. One example is modified IL-8 containing amino acid substitutions at selected positions.
Since the first chemokines and their receptors have been identified, the interest on exactly understanding their roles in normal and diseased physiology has become more and more intense. The constant need for new drugs with modes of action different from those of existing drugs support the development of new protein-based GAG-antagonists and their use in therapeutic applications, specifically for the prevention and treatment of cancer metastasis.
Although several proteins with increased GAG binding affinity and reduced receptor binding activity had been developed in the past, there is still need to develop proteins which show selective competition in GAG binding and thus can avoid negative side effects due to unselective binding affinity. Since for most of the GAG-binding proteins the exact binding epitope on the glycan is not known, targeting of such specific epitopes is still very challenging. It has been found in the past that engineering additional basic amino acids at many positions into a given GAG-binding protein can lead to the unwanted displacement of many proteins from a typical GAG co-receptor molecule on top of the target GAG-binding protein. In addition, oligomerisation of a GAG-binding protein can cause further unwanted and unspecific displacement reactions. Furthermore, therapeutic GAG-binding proteins should exhibit a serum half life which avoids daily dosing.